Detection of DNA amplification in droplets after polymerase chain reaction (PCR) has been demonstrated, mainly by use of fluorescent labels (e.g., probes, such as the Taqman probe) as markers for amplification events. As DNA is amplified, a fluorophore is released from its quenching molecule, and fluorescence can result when the sample is interrogated with an appropriate wavelength of light.
Despite the widespread use of this technique, there are several disadvantages to detecting reaction events via fluorescence, including the need for a fluorescent microscope and expensive optical equipment, the extra processing steps and expense required to incorporate fluorophore and quencher molecules into a probe, and the qualitative or inconsistent result that fluorescence measurements provide (e.g., as a result of photobleaching effects). In particular, photobleaching possesses the drawback of requiring normalization of fluorescent signals in order to quantify results. Alternative techniques for detection of nucleic acid amplification events include staining with an intercalating dye, such as ethidium bromide or SYBR Green. However, these materials are hazardous to work with and bind non-specifically to all double-stranded DNA.
Prior approaches using the concept of electrical impedance spectroscopy (EIS) have achieved high-sensitivity detection of nucleic acids and proteins by measuring direct binding events of molecules to the surface of an electrode. To induce binding, the electrode surface can be functionalized with a variety of materials, such as complementary nucleic acid strands, antibodies, or antigen molecules. Binding of a sufficient number of molecules of interest to the electrode surface is detectable as a change in the impedance of the system. However, conventional systems require static measurement, wherein molecules must be conjugated directly to the electrode surface. This is an inherently slow process, and one that is not amenable to high-throughput or continuous processing. Detection of binding events typically requires timescales on the order of tens of minutes. Further, multiplexed detection using fluorescence is limited by the number of available fluorescent probes, which is governed by the physical phenomenon of spectral overlap of these probes.